This invention relates to use of betel leaf extract to induce IFN-gamma production from human peripheral blood T cells.
Stinging nettle leaf extracts are registered in Germany for adjuvant therapy of rheumatic diseases. In a whole blood culture system, the nettle extract IDS 23 (Rheuma-Hek) inhibited lipopolysaccharide stimulated monocyte cytokine expression, indicating an immunomodulating effect. (Antirheumatic Effect of IDS 23, a Stinging Nettle Leaf Extract, on in vitro expression of T helper cytokines). The applicants investigated the immunomodulating effects of betel leaf extracts on phytohemagglutinin (PHA) stimulated peripheral blood mononuclear cells (PBMC) in vitro. Betel leaves has a strong pungent aromatic flavor and widely used as a masticatory. Generally, mature or over mature leaves, which has ceased growing but not yet become brittle are used for chewing. The basic preparation for chewing purposes consists of betel leaf smeared with hydrated lime and catechu to which scrapings of arecanut are added; flavorings such as coconut shavings, clove, cardamon, fennel, powdered liquorice, nutmeg and also tobacco are used according to one""s taste. In some places prepared pan is covered with silver or gold leaf. As a masticatory, it is credited with many properties: it is aromatic, digestive, stimulant and carminative. Medicinally it is useful in catarrhal and pulmonary affections; it is also used for poultices. The effects of chewing of betel with arecanut and other adjuncts are the excitation of the salivary glands and the irritation of the mucous membrane of the mouth. The red coloration produced is due to a pigment in the arecanut, which manifests itself under the action of alkali in lime and catechu. A mild degree of stimulation is produced, resulting in a sensation of warmth and well being, besides imparting a pleasant odor. The most important factor determining the aromatic value of the leaf is the amount and particularly the nature of the essential oil present. Betel leaves from different regions vary in smell and taste. The most pungent is the Sanchi type, while the most mild and sweet ones are from Madras. The betel leaves contain essential oils, the content of oil varies from 0.7 to 2.6 per cent depending upon the varieties of leaves. The oil consists of phenols and terpens. The higher the proportion of phenol oil, the better the quality. An isomer of eugenol named chavibetol (betel phenol; 4-allyl-2-hydroxy-1-methoxy benzene) is considered to be the characteristic constituent of betel oil. It is however, absent in Indian samples. Betel oil of Indian types contain as a predominant phenolic constituent. Oil of betel has been used in the treatment of various respiratory catarrhs, under as a local application either by gargle or by inhalation in diphtheria. It has carminative properties. It exhibits in different action on the central nervous system of mammals; lethal doses produce deep narcosis leading to death within a few hours. The essential oil and extracts of the leaves possess activity against several Gram-positive and Gram-negative bacteria such as Micrococcus pyogenes var. albus and var. aureus, Bacillus subtilis and B. megaterium, Diplococcus pneumoniae, Streptococcus pyogenes, Escherichia coli, Salmonella typhosa, Vibrio comma, Shigella dysenteriae, Proteus vulgaris, Pseudomonas solanacaerum, Sarcina lutea and Erwinia carotovora. The oil is found to be lethal in about 5 minutes to the protozoa Paramaecium caudatum (Wealth of India, Vol.8 pp.84-94). It inhibits the growth of Vibrio cholerae, Salmonella typhosum and Shigella flexneri and Escherichia coli. Steamxe2x80x94distillate of the leaves showed activity against Mycobacterium tuberculosis. 
The main object of the present invention is to provide a method for inducing IFN-xcex3 from human peripheral blood mononuclear cells.
Another object of the present invention relates to use of betel leaf extract for inducing IFN-xcex3 production from human peripheral blood T cells.
Another object of the invention is to provide a composition comprising betel leaf extract, which is useful as Th1 type immunomodulator.
To meet the above objects, the present invention provides a method for inducing IFN-xcex3 from human peripheral blood mononuclear cells. The invention also relates to use of betel leaf extract for inducing IFN-xcex3 production from human peripheral blood T cells.
Accordingly, the invention relates to a method for inducing IFNxcex3 from human peripheral blood mononuclear cells wherein the said method comprising preparing water extract of betel leaf; preparation of human peripheral blood mononuclear cells; incubation of hPBMC with betel leaf extract for a period of 18-48 hours; extraction of RNA for cytokine specific RT-PCR or for flow cytometry for the detection of intracellular cytokine protein; subjecting RNA for RT-PCR to obtain PCR products using IFNxcex3 specific known primers and enhancement of IFNxcex3 as reflected by IFNxcex3 specific band.
Alternatively, one more method for inducing IFNxcex3 produced from human peripheral blood mononuclear cells wherein the said process comprising subjecting incubated cells for intracellular staining for IFNxcex3; analysis of stained cells in flow cytometer; and enhancement of IFNxcex3 positive cells to at least seven fold.
A method for using betel leaf extract as Th1 type immuno-modulator wherein the said method comprising:
a) administering to a subject at least 5 to 10 mg/ml/kg body wt. of betel leaf extract, and
b) administering the extract through oral or intramuscular route once in a day for a period of at least one month,
In an embodiment of the present invention provides a pharmaceutical composition useful as Th1 type immunomodulator, said composition comprising an effective amount of betel leaf aqueous extract or lyophilized betel leaf extract together with or associated with an additive.
In still another embodiment of the invention, the additive is selected in such a manner that does not interfere with the activity of betel leaf extract.
Yet another embodiment of the invention, the additive is selected from nutrients such as proteins, carbohydrates, sugar, talc, magnesium stearate, cellulose, calcium carbonate, starch-gelatin paste and/or pharmaceutically acceptable carriers, excipient, diluent or solvent.
Yet another embodiment of the invention, the aqueous extract, lyophilized product or the composition is administered orally or intramuscularly.
Yet another embodiment of the invention, the oral route is in the form of capsule, syrup, concentrate, powder or granules.
Yet another embodiment of the invention, the ratio of betel leaf extract to the additive is in the range between 10-1:1-10.
Yet another embodiment of the invention, the betel leaf extract, lyophilized extract or the composition comprising the betel leaf extract is administered at a dosage level between 5 to 10 mg/kg of body weight at least once in a day for one month.
In one more embodiment of the present invention, a method of treating a subject to provide Th1 type immuno-mudulation, said method comprising administering a pharmaceutically effective amount of betel leaf extract, lyophilized extract or a composition comprising the extract to the subject.
Yet another embodiment of the present invention, the additive is selected in such a manner it does not interfere with the activity of lyophilized betel leaf extract.
Yet another embodiment of the invention, the additive is selected from nutrients such as proteins, carbohydrates, sugar, talc, magnesium stearate, cellulose, calcium carbonate, starch-gelatin paste and/or pharmaceutically acceptable carriers, excipient, diluent or solvent.
Yet another embodiment of the invention, the aqueous extract, lyophilized product or the composition is administered orally or intramuscularly.
Yet another embodiment of the invention, the oral route is in the form of capsule, syrup, concentrate, powder or granules.
Yet another embodiment of the invention, the ratio of betel leaf extract to the additive is in the range between 10-1:-10.
Yet another embodiment of the invention, the betel leaf extract, lyophilized extract or the composition comprising the betel leaf extract is administered at a dosage level between 5 to 10 mg/kg of body weight at least once in a day for one month.
In one more embodiment of the present invention, use of betel leaf extract in association with suitable carriers/additive as Th1 type immunomodulator.
Yet another embodiment of the present invention, the additive is selected in such a manner it does not interfere with the activity of lyophilized betel leaf extract.
Yet another embodiment of the invention, the additive is selected from nutrients such as proteins, carbohydrates, sugar, talc, magnesium stearate, cellulose, calcium carbonate, starch-gelatin paste and/or pharmaceutically acceptable carriers, excipient, diluent or solvent.
Yet another embodiment of the invention, the aqueous extract, lyophilized product or the composition is administered orally or intramuscularly.
Yet another embodiment of the invention, the oral route is in the form of capsule, syrup, concentrate, powder or granules.
Yet another embodiment of the invention, the ratio of betel leaf extract to the additive is in the range between 10-1:1-10.
Yet another embodiment of the invention, the betel leaf extract, lyophilized extract or the composition comprising the betel leaf extract is administered at a dosage level between 5 to 10 mg/kg of body weight at least once in a day for one month.
One more embodiment of the present invention relates to the preparation of betel leaf extracts comprising the following steps;
1) washing of the fresh leaves of Piper betel and homogenizing in a mixture blender;
2) sonicating in an ultrasonic bath with 2 to 3 bursts each for 15 minutes and filtering the extract, if desired repeating the extraction at least once and drying; and
3) lyophilizing the extract to get a semi-solid mass
In one more embodiment of the invention, the lyophilized extract is obtained by freeze drying the aqueous extract by conventional methods.
In another embodiment, the water extract is prepared from following types of betel leaf (Piper betel) namely Wild type, Climber type, Bangla type and Sweet type.